Dual Intracellular and Extracellular Expression Technology (dIEE) for Parkinson?s Disease Abstract Alpha-synuclein (?-syn) is a key player in the pathogenesis of Parkinson's Disease (PD), a devastating neurodegenerative illness that will afflict over 920,000 Americans by 2020. Oligomers and aggregates of this presynaptic-vesicle-associated protein characterize major pathological findings in PD that correlate well with progressive motor neuron functional decline. While ?-syn is normally localized intracellularly at synapses, during PD, oligomeric ?-syn can spread damage extracellularly in a defined path through the brain in a prion-like manner, where the altered conformation of oligomeric ?-syn catalyzes creation of increased aberrant ?-syn. Pathological ?-syn then creates at least three challenges for a potential therapeutic: a) block intracellular initiation-activity, b) clear intracellular pathological ?-syn, and c) clear toxic extracellular ?-syn to prevent progression. To address these problems, we have designed a recombinant adeno-associated viral (AAV) vector to achieve long-term, stable central nervous system (CNS) delivery of an antibody. This vector will simultaneously deliver an intracellular anti-oligomeric ?-syn single-chain intrabody (iAb) combined with a secreted extracellular anti-?-syn N-Terminal antibody (sAb). Our novel dual intrabody/antibody approach employs proprietary dual Intracellular and Extracellular Expression technology (dIEE). We hypothesize that the combined effect of the anti-?-syn intrabody and secreted antibody will reduce alpha-synuclein-mediated pathology with concomitant improvement of PD. In addition, AAV delivery addresses the blood brain barrier (BBB) problem of systemic antibody therapy and represents a novel and potentially practical approach for the treatment of additional neurodegenerative diseases. In Phase I, we will identify potent anti-oligomeric human ?- syn antibodies, construct the vectors, and test their expression in vitro and in the brains of recipient mice. We are optimistic that our AAV dual expression system for simultaneous delivery of anti-oligomeric-?-syn intrabodies and antibodies to the brains of affected patients will provide a novel restorative therapy for PD.